We thank Osman Kaftanoglu for helpful advice and assistance during filming. We would like to thank the anonymous reviewers for insightful comments. This work was supported by the Research Council of Norway (grants 180504, 191699, and 213976), Marie Curie/FP7 (project ref. 238665), the National Institute on Aging (grant NIA P01 AG22500), and the Pew Charitable Trusts.

1. Decoupling Senescence from Chronological Age
This section describes the setup of double cohort colonies, which consist of a cohort of identified individuals that share the same chronological age ("single age cohort") and a cohort of nest bees. Same aged individuals of the single age cohort will eventually separate into different worker-types with different aging dynamics - these are nurse bees with slowed and forager bees with accelerated functional decline. All procedures are described for one experimental colony. We advise, however, to perform experiments for at least two colony replicates so that colony effects can be controlled for (two-replicate-design).
<ol>
	<li>Preparing hive boxes for the double cohort colonies: Prepare one regular hive box that receives two food combs with honey, another food comb with pollen, and two empty combs. Make sure to locate a mated queen as well as a donor colony with more than 3,000 nest bees. Both will be introduced later (1.3).</li>
	<li>Obtaining and marking individuals with similar chronological age: Collect combs with sealed brood that is about to emerge. For one replicate expect to collect combs with a total of 3,000-5,000 capped brood cells. For each replicate use a balanced amount of brood from at least three different hive sources to avoid skewed distributions of maternal genotypes (hive origin).
	<ol>
		<li>Place brood combs in an incubator set to 34 &deg;C with 60-70% relative humidity. Make sure to store combs in such a way that emerging brood cannot escape.</li>
		<li>Let bees emerge for two days and mark these bees with a small paint tag on the thorax (e.g. Uni POSCA, Mitsubishi Pencil Co. Ltd.). The paint mark will allow identifying the bees of the single age cohort (day of emergence), and to distinguish them from other replicate colonies.</li>
	</ol>
	</li>
	<li>Setting up a double cohort colony, which includes the cohort of identified, single age bees: On the day young bees have been marked, collect about 2,500-3,000 nest bees from a donor colony (compare section 1. in Discussion), and add these unmarked bees to the hive box that was prepared before (see step 1.1). The latter individuals will constitute the unidentified nest bee cohort.
	<ol>
		<li>Add the queen, which will be initially confined to a queen cage (commercially available). Seal the cage with edible candy (e.g. Apifonda, S&uuml;dzucker AG, Mannheim/Ochsenfurt, Germany) to make worker bees slowly release the queen.</li>
		<li>Add the newly emerged and marked bees, which will constitute the single age cohort. These bees are the only marked individuals, and are the focus group for all the following steps.</li>
	</ol>
	</li>
	<li>Monitoring foraging onset and marking foragers: To assess onset and dynamics of the nurse-to-forager transition in the single age cohort, monitor the demographical development of foraging activity every other day for each colony. Begin counting five days after colonies were set up (Figure 1).
	<ol>
		<li>Count the total number of bees returning from foraging flights (entrance counts) within 3 x 20 min observation periods at fixed times. Make sure to not count bees during periods of orientation flights (see Discussion).</li>
		<li>When entrance counts indicate considerable foraging activity to commence (&gt;100 entrance counts/day), begin marking foragers. To do so, foragers of the single age cohort (single marked individuals) receive a second paint mark upon returning from their first foraging flights. This paint mark will specify the day of foraging onset, and will allow to later identify the foraging age for each forager.</li>
		<li>Repeat daily markings until a sufficient number of bees has been marked. For estimating a sufficient number of marked foragers, expect a retrieval rate of no more than 5-10% after these bees had been aged, typically after 14 days of foraging.</li>
	</ol>
	</li>
	<li>Sampling: Since all initially marked bees have a similar chronological age, age-matched groups of nurses and old foragers can be collected simultaneously, when foragers have foraged for &ge;14 days.
	<ol>
		<li>Single marked nurse bees are collected within the hive, and are identified by nursing behavior (feeding and cleaning of larvae with heads put down in open brood cells).</li>
		<li>Double marked foragers are also collected within the hive before daily foraging activity begins.</li>
		<li>Collect bees in cages (tubes, boxes) that provide sufficient ventilation, and keep dark until further processing. Alternatively, for transcriptomic, epigenetic or proteomic studies, directly snap freeze bees in liquid nitrogen. Collect balanced numbers of individuals from all test groups and replicate colonies.</li>
	</ol>
	</li>
</ol>
2. Reversal of Workers with Rapid to Workers with Slowed Aging by Changing the Hive's Demography
This section details how the reversal from workers with accelerated aging (foragers) to workers with slowed aging (nurse bees) is performed. Such behavioral reversal is induced, when foragers experience a lack of nurse bees, which normally engage in brood care. The reversion procedure will separate a single colony replicate into two hives: one hive with the nurse bee fraction ("nurse-derived"), and another one with the forager fraction ("forager-derived"). After successful reversal, possible symptoms of plastic and reversed aging can be studied in the single age cohort with reverted workers, continuing foragers, continuing nurse bees and newly recruited foragers. As before, identified bees of the single age cohort, not the cohort of unidentified nest bees, constitute the experimental focus group.
<ol>
	<li>Preparation: Replicate hives with nurses (single marked) and foragers (double marked) are made available as described in the previous section. Make sure to not begin reversion with less than 500 marked foragers per replicate colony to ensure sufficient retrieval after the reversion has been completed.
	<ol>
		<li>For safe identification of test groups after reversal it is crucial that the entire forager population in the original hive has been marked off before reversion. The following procedure is described for one replicate.</li>
		<li>The day before reversion, prepare one additional hive box for the forager-derived hive (see step 1.1). Locate two queens and two brood combs from donor hives. Before transfer to the experimental colonies brush away all adult bees from these combs. One caged queen (see step 1.3) and one brood comb will replace queen and brood combs in the original hive box. The other set of queen and brood comb will be used the next day for the new hive box. Similar allocation of new brood combs and alien queens for both, the original and the new hive is advised to make sure that separated foragers and nurses will equally experience changed hive cues ("hive smell").</li>
	</ol>
	</li>
	<li>Reversion: In the morning, just before the reversion, add the caged queen and the brood comb to the new box that will receive the forager-derived fraction. Wait until the peak foraging time begins. Then move the original colony with marked foragers and nurse bees at least 100 m away from the original location.
	<ol>
		<li>At the original location, set up the new box for the forager-derived hive with brood and queen only.</li>
		<li>Foragers will leave the dislocated original hive box, and head back to the original location. Allow foragers to return to the original location for 2 hr in order to achieve next to complete separation of the forager population from nest bees.</li>
		<li>Then, to terminate separation, close off the original, now "nurse-derived" hive, and transfer it to an apiary at least 3 km away.</li>
	</ol>
	</li>
	<li>Hive maintenance and monitoring for successful social task reversal: Check the experimental hives regularly for healthy, open brood.
	<ol>
		<li>During the first days after colony manipulation replace unattended and dead brood to reduce potential pathogen load.</li>
		<li>To validate successful reversal within the forager-derived hive, take pictures of brood combs before introducing these, and again when combs are replaced or when the reversion experiment is completed (Figure 2). Areas with previously uncapped brood and with open, live brood are reliable markers of nursing activity in forager-derived colonies.</li>
	</ol>
	</li>
	<li>Sampling: Physiological effects that accompany social reversal can be detected 3-8 days after foragers and nurses were separated.
	<ol>
		<li>We advise sampling all test groups, i.e. reverted workers and continuing foragers (forager-derived hive), as well as continuing nurses and newly-recruited foragers (nurse-derived hive) 8 days after reversal is initiated.</li>
		<li>Collect samples as described in step 1.5.</li>
	</ol>
	</li>
</ol>
3. Analyzing Worker-type Specific Cellular Senescence Patterns by Quantification of Lipofuscin
Lipofuscin is a universal biomarker of cellular senescence. As an intrinsic accumulation product, lipofuscin's specific autofluorescence (emissionmax = 530-650 nm) can be used for detection18.
<ol>
	<li>Dissection and fixation: Chill bees on ice until motionless; remove and dissect out the desired tissue sample.
	<ol>
		<li>Transfer into fixative (4% paraformaldehyde in phosphate-buffered saline, PBS; pH 7.2) for overnight incubation at 4 &deg;C.</li>
		<li>Wash samples 3 times in PBS.</li>
	</ol>
	</li>
	<li>Tissue processing and mounting: Cut tissue samples into sections with no more than 40 &mu;m thickness, for example by using a vibrating blade microtome, e.g. Leica VT 1000S (Leica Biosystems, Nussloch, Germany).
	<ol>
		<li>For clearing, incubate tissue sections overnight in 30% glycerol (PBS), and another 2 hr in 50% glycerol (PBS).</li>
		<li>Mount sections on microscopic slides in 50% glycerol (PBS). For long-term storage seal cover slips with nail polish.</li>
	</ol>
	</li>
	<li>Image acquisition: To detect lipofuscin, we suggest using a laser scanning confocal microscope that provides laser lines with &lambda; = 514, 561nm or similar for excitation, and with the detector bandwidth set to 570-650 nm.
	<ol>
		<li>For better identification of lipofuscin, include a second channel, and do a simultaneous scan at shorter wavelength spectra (excitation = 405 nm; emission = 410-450 nm). The longer wavelength channel will reveal both, granular lipofuscin, but also unspecific "background" due to autofluorescent trachea and other non-granular structures. The second, shorter wavelength channel will only reveal unspecific autofluorescence, but not lipofuscin. Thus, lipofuscin identification can be facilitated by comparing the signals in both channels, with only one of them revealing the granules with lipofuscin specific fluorescence.</li>
		<li>For acquisition of high-resolution images use an objective with 40X magnification or higher, and preferably a numerical aperture of 1.25 or higher. Scan image stacks with dimensions of about 100 x 100 x 10 &mu;m3. Every individual and tissue sample has to be represented by multiple image stacks.</li>
		<li>To reduce intra-individual and inter-individual variation caused by technical variation, always keep laser power and detector sensitivity constant.</li>
		<li>To reduce bias by day-to day technical variations scan equal sample numbers of all test groups in each of the several scanning sessions.</li>
	</ol>
	</li>
	<li>Image processing: Use software packages with modules that allow for advanced processing of microscopic image stacks, for example ImageJ (U.S. National Institutes of Health, Bethesda, Maryland, USA, <a href="http://imagej.nih.gov/ij/" target="_blank">http://imagej.nih.gov/ij/</a>).
	<ol>
		<li>Generate a 2D maximum projection for each of the 3D image stacks.</li>
		<li>Apply a Gauss filter with a modest kernel size in order to attenuate high frequency noise, and to preserve structures with dimensions of lipofuscin granules.</li>
		<li>Merge both color channels to facilitate identification of lipofuscin (see step 3.2).</li>
	</ol>
	</li>
	<li>Image analyses: Make sure that the subject performing the quantification steps will be blind to test group identity.
	<ol>
		<li>For all images, first choose a region of interest (ROI) that covers the relevant structures, and has similar dimensions as the ROIs from other images.</li>
		<li>Then select the desired number of lipofuscin granules that represent each ROI. When selecting lipofuscin granules within a ROI, the application of the following rules will reduce subjective bias.
		<ol>
			<li>Choose a consistent location for selecting the first granule. This can be, for example, the leftmost edge of a ROI, and the granule that is closest to the edge will always be the first selection.</li>
			<li>One after another, granules are chosen that are closest to the previous selection (next neighbor).</li>
			<li>When choosing the next neighbor, move only into one direction, for example only search right from the previous selection. This rule prevents that the selection is dominated by occasional clusters of densely packed granules.</li>
			<li>When selection is completed, assess the size of each lipofuscin particle by outlining and measuring the respective granule area. Use appropriate statistical tests to compare individuals of the different test groups.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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We have nothing to disclose.

We here adopt previously described approaches8,16,17,19,20, and integrate them into a single workflow that will facilitate studying flexible aging in honey bees. Our aim is to provide scientists that are novice to this field with a standardized tool set to obtain relevant sample material, and to improve experimental reproducibility among different research teams. While our procedures are simplified and do not require special equipment as in earlier descriptions (compare for example8), some measures ...

The complex colony structures of highly social animals are maintained through the interaction of a reproductive caste, and a helper caste of typically non-reproducing workers with different social task behaviors. In the different workers, specific physiological adaptations enable distinct sib care behaviors, and are also linked to extreme lifespan differences. Honey bees and mole rats represent the best-developed animal models to study how sociality is linked to patterns of accelerated, negligible or reversed aging1-3.
In honey bee colonies, a single egg-laying queen is assisted by thousands of workers that tend the brood, forage for food, and engage in guarding, thermoregulation or hygienic behaviors4. Among these workers are the extremely short-lived foragers, nurse bees with intermediate, and winter (diutinus) bees with longest lifespans. Individuals, however, are not permanently bound to a certain worker-type, but display a flexible behavioral ontogeny: they change from one social task behavior to another ("temporal castes"). Callow bees can change to brood tending nurse bees, which eventually may change to outside foraging. However, callow nest bees can also transform into longest-lived winter bees, and short-lived foragers can even revert into typically longer-lived nurses. Workers with extreme (winter bees) and intermediate (nurse bees) lifespan have well-developed food production and storage organs with copious resources - as opposed to short-lived foragers (reviewed in1,5). However, that the regulation of individual lifespan goes beyond simple changes in an individual's resource balance is suggested by research on a yolk protein, which has diverse adapted functions in the non-reproducing worker caste, such as jelly production6, hormonal control7, immune8 and anti-oxidant defense9.
Patterns of functional decline (senescence) mirror lifespan disparities among workers, as established for olfactory, and also for other brain or motor functions10-13. Specifically, the significant decline in learning function after only two weeks of foraging matches a similar mortality progression in foragers14, as opposed to the lack of detectable decline (negligible senescence) in long-lived winter bees15.
To identify the molecular fingerprints of flexible aging we adapted established experimental paradigms that allow for monitoring and manipulating aging-type transitions8,16,17. Experiment 1 details how to obtain samples in which the effects of chronological age and worker-type specific social behaviors on aging can be separated. Experiment 2 describes the reversal of foragers with accelerated into nurse bees with slowed aging dynamics. Experiment 3 provides an approach for probing effects of cellular senescence by anatomical quantification of an established biomarker for cellular aging (lipofuscin)18.

<table border="1">
 <tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Apifonda</td>
 <td>S&uuml;dzucker AG, Mannheim/Ochsenfurt, Germany</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>paraformaldehyde</td>
 <td>Sigma-Aldrich</td>
 <td>158127</td>
 <td></td>
 </tr>
 <tr>
 <td>phosphate-buffered saline</td>
 <td>Sigma-Aldrich</td>
 <td>P4417</td>
 <td></td>
 </tr>
 <tr>
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obtaining specimens with slowed, accelerated and reversed aging in the honey bee model

Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best established model to study how plasticity in lifespan and aging is explained by social factors.
The worker caste of honey bees includes nurse bees, which tend the brood, and forager bees, which collect nectar and pollen. Previous work has shown that brain functions and flight performance senesce more rapidly in foragers than in nurses. However, brain functions can recover, when foragers revert back to nursing tasks. Such patterns of accelerated and reversed functional senescence are linked to changed metabolic resource levels, to alterations in protein abundance and to immune function. Vitellogenin, a yolk protein with adapted functions in hormonal control and cellular defense, may serve as a major regulatory element in a network that controls the different aging dynamics in workers.
Here we describe how the emergence of nurses and foragers can be monitored, and manipulated, including the reversal from typically short-lived foragers into longer-lived nurses. Our representative results show how individuals with similar chronological age differentiate into foragers and nurse bees under experimental conditions. We exemplify how behavioral reversal from foragers back to nurses can be validated. Last, we show how different cellular senescence can be assessed by measuring the accumulation of lipofuscin, a universal biomarker of senescence.
For studying mechanisms that may link social influences and aging plasticity, this protocol provides a standardized tool set to acquire relevant sample material, and to improve data comparability among future studies.

Protocol sections 1 and 2 detail how test groups can be obtained to study attributes of accelerated, slowed and reversed aging in colonies with a single age cohort. To monitor worker-type differentiation that accompanies the normal ontogeny we assessed forager counts ("entrance counts") for 6 colonies (Figure 1, compare section 1). The graphs show that considerable change from nurse to the forager state is typically not observed before individuals are more than 10 days old. Marked variability in forager ...

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Obtaining Specimens with Slowed, Accelerated and Reversed Aging in the Honey Bee Model

In honey bee workers, aging depends on social behaviors rather than on chronological age. Here we show how worker-types with very different aging patterns can be obtained and analyzed for cellular senescence.

Societies of highly social animals feature vast lifespan differences between closely related individuals. Among social insects, the honey bee is the best ...

obtaining-specimens-with-slowed-accelerated-reversed-aging-honey-bee

Research

JoVE Journal

Biology

11.2K 조회수.  Norwegian University of Life Sciences.  꿀벌 노동자, 노화는 사회적 행동보다는 연대기 나이에 따라 달라집니다. 여기에서 우리는 매우 다른 노화 패턴과 노동자 유형을 얻을과 세포 노화를 분석 할 수있는 방법을 보여줍니다. 

비디오: Obtaining Specimens with Slowed, Accelerated and Reversed Aging in the Honey Bee Model

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to measure odor evoked activity in the antennal lobe1. From then on, it has been continuously refined and applied to different neuropiles in the bee brain. Here, we describe the preparation currently used in the lab to measure activity in mushroom body neurons using a dextran coupled calcium-sensitive dye (Fura-2). We retrogradely stain mushroom body neurons by injecting dye into their axons or soma region. We focus on reducing the invasiveness, to achieve a preparation in which it is still possible to train the bee using PER conditioning. We are able to monitor and quantify the behavioral response by recording electro-myograms from the muscle which controls the PER (M17)2.
After the physiological experiment the imaged structures are investigated in greater detail using confocal scanning microscopy to address the identity of the neurons.

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

Bees can be conditioned in an appetitive olfactory learning paradigm (PER-conditioning). Using odors as stimuli, we established a method in which behavior is recorded while simultaneously Calcium Imaging is used to measure odor evoked activity in mushroom body neurons in vivo.

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees collect and convert to beebread by placing colonies in a specially constructed enclosed flight area. Methods were developed to analyze the protein and amino acid composition of the pollen and beebread. We also describe how consumption of the beebread was measured and methods used to determine adult worker bee hemolymph protein titers after feeding on beebread for 4, 7 and 11 days after emergence. Methods were applied to determine if genotype affects the conversion of pollen to beebread and the rate that bees consume and acquire protein from it. Two subspecies (European and Africanized honey bees; EHB and AHB respectively) were provided with the same pollen source. Based on the developed methods, beebread made by both subspecies had lower protein concentrations and pH values than the pollen. In general, amino acid concentrations in beebread made by either EHB or AHB were similar and occurred at higher levels in beebread than in pollen. Both AHB and EHB consumed significantly more of the beebread made by AHB than by EHB. Though EHB and AHB consumed similar amounts of each type of beebread, hemolymph protein concentrations in AHB were higher than in EHB. Differences in protein acquisition between AHB and EHB might reflect environmental adaptations related to the geographic region where each subspecies evolved. These differences could contribute to the successful establishment of AHB populations in the New World because of the effects on brood rearing and colony growth.

Here are methods to quantify nutrients in pollen before and after its conversion to beebread by two subspecies of honeybees. We describe techniques to measure beebread consumption and resulting protein titers in both subspecies.

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees ...

Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function

The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination services, as well as an important model social insect used to study memory and learning, aging, and more. Here we describe a detailed protocol for the dissection of the dorsal abdominal wall of a bee in order to visualize its dorsal vessel, which serves the role of the heart in the insect. A successful dissection will expose a functional heart that, under the proper conditions, can maintain a steady heartbeat for an extended period of time. This allows the investigator to manipulate heart rate through the application of cardiomodulatory compounds to the dorsal vessel. By using either a digital microscope or a microscope equipped with a digital camera, the investigator can make video recordings of the dorsal vessel before and after treatment with test compounds. The videos can then be scored at a time convenient to the user in order to determine changes in heart rate, as well as changes in the pattern of heartbeats, following treatment. The advantages of this protocol are that it is relatively inexpensive to set up, easy to learn, requires little space or equipment, and takes very little time to conduct.

The abdominal dorsal vessel of the honey bee and other insects serves as the functional equivalent of the mammalian heart and plays an important role in nutrient transport, waste removal, immune function, and more. Here we describe a protocol for the visualization and pharmacological manipulation of bee heart rate.

The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination ...

Monitoring Colony-level Effects of Sublethal Pesticide Exposure on Honey Bees

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment methods. Here we describe methods to measure the quantities of adult bees, brood, and food resources by weighing hives and hive parts, by photographing frames, and by installing hives on scales and with internal sensors. Data from these periodic evaluations are then combined with running average and daily detrended data on hive weight and internal hive temperature. The resulting datasets have been used to detect colony-level effects of imidacloprid applied in a sugar syrup as low as 5 parts per billion. The methods are objective, require little training, and provide permanent records in the form of sensor output and photographs.

Sublethal doses of pesticides may affect colonies in ways that are difficult to detect using only periodic or visual methods. Methods to measure total adult bee mass, brood, and food resources by weighing hives and hive parts, photographing frames, and installing sensors, are provided. Data analysis is also addressed.

The effects of sublethal pesticide exposure to honey bee colonies may be significant but difficult to detect in the field using standard visual assessment ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

SA-&amp;#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...

A 3D Printed Pollen Trap for Bumble Bee (Bombus) Hive Entrances

To verify the plant sources from which bumble bees forage for pollen, individuals must be collected to remove their corbicular pollen loads for analysis. This has traditionally been done by netting foragers at nest entrances or on flowers, chilling the bees on ice, and then removing the pollen loads from the corbiculae with forceps or a brush. This method is time and labor intensive, may alter normal foraging behavior, and can result in stinging incidents for the worker performing the task. Pollen traps, such as those used on honey bee hives, collect pollen by dislodging corbicular pollen loads from the legs of workers as they pass through screens at the nest entrance. Traps can remove a large quantity of pollen from returning forager bees with minimal labor, yet to date no such trap is available for use with bumble bee colonies. Workers within a bumble bee colony can vary in size making size selection of entrances difficult to adapt this mechanism to commercially reared bumble bee hives. Using 3D printing design programs, we created a pollen trap that successfully removes the corbicular pollen loads from the legs of returning bumble bee foragers. This method significantly reduces the amount of time required by researchers to collect pollen from bumble bee foragers returning to the colony. We present the design, results of pollen removal efficiency tests, and suggest areas of modifications for investigators to adapt traps to a variety of bumble bee species or nest box designs.

A 3D Printed Pollen Trap for Bumble Bee Bombus Hive Entrances

We present a non-lethal and automated mechanism to collect pollen from bumble bee (Bombus) workers returning to a hive. Instructions for producing, preparing, installing and using the devices are included. By using 3D-printed objects, modification to the design was timely, efficient and allowed for quick turnaround for testing.

To verify the plant sources from which bumble bees forage for pollen, individuals must be collected to remove their corbicular pollen loads for analysis.  ...

Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka (Oryzias latipes)

Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in all vertebrates by providing feedback to the brain and pituitary. Teleost fishes possess a higher degree of tissue plasticity and variation in reproductive&#160;strategies compared to mammals and appear to be useful models to investigate the role of sex steroids and the mechanisms by which they act. The removal of the main source of sex steroid production using gonadectomy together with blood sampling to measure steroid levels has been well-established and fairly feasible in bigger fish and is a powerful technique to investigate the role and effects of sex steroids. However, these techniques raise challenges when implemented in small size teleost models. Here, we describe the step-by-step procedures of gonadectomy in both males and female Japanese medaka followed by blood sampling. These protocols are shown to be highly feasible in medaka indicated by a high survival rate, safety for the life span and phenotype of the fish, and reproducibility in terms of sex steroid clearance. The use of these procedures combined with the other advantages of using this small teleost model will greatly improve the understanding of feedback mechanisms in the neuroendocrine control of reproduction and tissue plasticity provided by sex steroids in vertebrates.

Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka Oryzias latipes

The article describes a quick protocol to gonadectomize and sample blood from the small teleost fish, using Japanese medaka (Oryzias latipes) as a model, to investigate the role of sex steroids in animal physiology.

Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in ...

Collection and Identification of Pollen from Honey Bee Colonies

Researchers often collect and analyze corbicular pollen from honey bees to identify the plant sources on which they forage for pollen or to estimate pesticide exposure of bees via pollen. Described herein is an effective pollen-trapping method for collecting corbicular pollen from honey bees returning to their hives. This collection method results in large quantities of corbicular pollen that can be used for research purposes. Honey bees collect pollen from many plant species, but typically visit one species during each collection trip. Therefore, each corbicular pollen pellet predominantly represents one plant species, and each pollen pellet can be described by color. This allows the sorting of samples of corbicular pollen by color to segregate plant sources. Researchers can further classify corbicular pollen by analyzing the morphology of acetolyzed pollen grains for taxonomic identification. These methods are commonly used in studies related to pollinators such as pollination efficiency, pollinator foraging dynamics, diet quality, and diversity. Detailed methodologies are presented for collecting corbicular pollen using pollen traps, sorting pollen by color, and acetolyzing pollen grains. Also presented are results pertaining to the frequency of pellet colors and taxa of corbicular pollen collected from honey bees in five different cropping systems.

We describe methods for collecting corbicular pollen from honey bees as well as protocols for color sorting, acetolysis, and microscope slide preparation of pollen for taxonomic identification. In addition, we present pellet color and taxonomic diversity of corbicular pollen collected from five cropping systems using pollen traps.

Researchers often collect and analyze corbicular pollen from honey bees to identify the plant sources on which they forage for pollen or to estimate ...